1. Field of the Invention
The present invention relates to an improved test composition for use in radioimmunoassays for the determination of haptens and antigens (hereinafter referred to as "ligands") in liquid samples including body fluids such as serum. The invention also concerns a method for preparing such test composition and an improved radioimmunoassay method and test kit.
Radioimmunoassay methods, in general, are based on the competition between a specific native ligand, the amount of which is to be determined in a sample, and a known amount of the same ligand in radioactively labelled form, for a limited number of available binding sites on an antibody which is specific towards the ligand under assay. Thus, in a system consisting of an unknown amount of unlabelled native ligand, a known amount of a radioactively labelled ligand and a limited known amount of antibody, the greater the concentration of unlabelled native ligand from the sample, the less the labelled ligand will be bound by the antibody.
If the concentration of labelled ligand and antibody is fixed and the only variable is the level of unlabelled ligand, it becomes possible to establish an assay system for measuring the unknown level of unlabelled ligand, by separating the ligand-antibody complex (the "bound" species) from the remaining free ligand (both labelled and unlabelled--the "free" species). The radioactivity of the unknown is compared with the values given by a range of known amounts of the ligand treated in the same manner. The values obtained from the determination of the standard samples may be used for establishing a standard calibration curve for the specific system and this curve may then be used to determine an unknown concentration of the ligand in an unknown sample.
There are many procedures for separating the ligand-antibody complex from the free unbound ligand, amongst which chromatoelectrophoresis, ascending paper-wick chromatography, precipitation by salts, organic materials or solvents, selective adsorption on various so-called "immunosorbents," either in suspension or on chromatographic columns, and ion exchange techniques may be mentioned as examples of such procedures and reference is made to the detailed discussions of these known methods in U.S. patent application Ser. No. 852,105, incorporated herein by reference filed Nov. 16, 1977 and assigned to the instant assignee.
The first step of most current immunoassay procedures involves the preparation of an incubation mixture (or reaction mixture) including a known quantity of the ligand under assay in radioactively labelled form, a known volume of the sample (i.e., an aqueous solution of the ligand under assay in native form), and a predetermined quantity of the specific binder for said ligand, which, as a rule, is an antibody. This reaction mixture is then incubated for a suitable time to cause a reaction between the ligand and the specific binder therefor so that a part of the ligand forms a ligand-binder complex. The preparation of this reaction mixture involves the dispensing of predetermined volumes of at least three different solutions and in many cases the number is even greater, e.g., when a buffer solution has to be added. Furthermore, in most clinical radioimmunoassays where the sample is a body liquid taken from a patient, the volumes involved are very small which renders their accurate measurement and dispensing a rather delicate and complicated task. It is thus easy to understand why the accuracy of radioimmunoassay methods is comparatively low, some of the major sources of inaccuracy being the errors in the micro-dispensing of the solutions of the components of the reaction mixture.
2. Description of the Prior Art
In order to increase the accuracy of radioimmunoassay methods as well as the shorten and simplify the experimental procedure performed by the medical laboratory technicians, it has been proposed to prepare the reaction mixture (or incubation mixture) in vessels already containing the required pre-measured amount of either the radioactively labelled ligand under assay or the specific binder therefor. Kits for specific immunoassay determinations comprising a number of test tubes each containing a pre-measured quantity of either of the above-mentioned components are commercially available. For better storage stability and convenient transportation, the test tubes in these kits contain the radioimmunoassay component in lyophilized form and the kit usually includes also a suitable buffer solution for dissolving (reconstituting) the lyophilized component in the test tubes before the other reactants are added. Although the amount of buffer solution, used for the reconstitution of the lyophilized component in the test tube, does not have to be measured with a high degree of accuracy, the use of kits of this type still requires the accurate measurement, usually by pipetting, of at least two solutions by the medical laboratory technician. Thus, if the lyophilized component in the test tubes is the radioactively labelled ligand, it is necessary to add, in addition to the above-mentioned buffer solution, accurate amounts of both the specific binder and of the sample to be assayed.
Some suggestion has been made to lyophilize both the radiolabelled ligand and the binder in the same container as exemplified by the disclosures in U.S. Pat. No. 4,017,597; British Pat. No. 1,411,381 and Belgian Pat. No. 852,990. However, the hitherto disclosed methods require careful layering of the radiolabeled ligand and the binder or instantaneous freezing of a mixture with no assurance that some complexing between the labelled ligand and the binder does not take place during the preparative process, during storage, or after reconstitution but before addition of the sample.
U.S. Pat. No. 4,107,284 discloses a stabilized radioimmunoassay reagent wherein the radiolabelled ligand and the binder are in the form of their complex. Upon reconstitution and use in an assay, a substantial incubation time will be required to permit dissociation for competition between labelled ligand and sample or native ligand.
Thus, there is a need for developing a stabilized, dry radioimmunoassay reagent wherein the labelled ligand and the antibody are not bound in the form of their complex. Such a reagent would provide a single reagent for introducing both the label and the antibody to the reaction mixture in their free, or non-complexed, forms.